CN102796808B - Methylation high-flux detection method - Google Patents
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Abstract
本发明提供一种甲基化高通量检测方法，特别提供了一种结合序列捕获和重亚硫酸盐测序的方法，该方法能够同时准确有效地分析几个样品中的目标区域甲基化状态，降低探针设计的难度，增加操作及应用的可行性，使得对全基因组内感兴趣的目标序列和区域进行高通量高精度的甲基化检测成为现实，并且该方法还具有针对性强以及节约成本和时间的特点。The present invention provides a high-throughput methylation detection method, especially a method combining sequence capture and bisulfite sequencing, which can accurately and effectively analyze the methylation status of target regions in several samples at the same time , reduce the difficulty of probe design, increase the feasibility of operation and application, and make high-throughput and high-precision methylation detection of target sequences and regions of interest in the whole genome a reality, and this method is also highly targeted As well as cost and time saving features.
Description
Technical field
The present invention relates to high-throughput genomic methylation DNA detection field, a kind of method that binding sequence is caught and bisulfite (bisulfite) checks order is particularly provided.In addition, the invention still further relates to the order-checking of exon group and the methylation analysis sequencing technologies field based on bisulfite conversion.The invention still further relates to the label that methylates (Index) joint technique, can realize and on a chip, carry out catching of several samples simultaneously.Method of the present invention is specially adapted to s-generation sequencing technologies, especially solexa sequencing technologies.
Background technology
DNA methylation is epigenetics research field one of the direction of focus the most, also becomes just gradually that Mammals grows and the epigenetics mark of the various diseases such as cancer.DNA methylation is not only modified chromatin Structure, genome stability has vital role, and in eukaryote, DNA methylation participates in regulate several biological processes, as fetal development, genomic imprinting, x chromosome inactivation, the adjusting of genetic expression and silence, generation (the Brena RM et al.2006 of the various diseases such as silence and mammal tumor of retrotransposon; Egger G et al.2004; Gu H et al.2010).DNA methylation biomarker is that the early stage assessment of various diseases comprises the detection assessment to high-risk individuality, and a large amount of reference informations are provided.
At present, the order-checking of exon group and the methylation analysis order-checking based on bisulfite conversion are all ripe.Take Illumina Solexa, AB SOLiD and Roche 454 as the s-generation sequencing technologies of representative had been rapidly developed in recent years, become the important tool of genomics research.The feature of s-generation sequencing technologies maximum is high-throughput, it can check order to hundreds of millions of DNA fragmentations simultaneously, a current high-flux sequence instrument once can produce the data up to 300Gb, be equivalent to a people's genome sequencing 100 times, high-flux sequence is, by ultrasonic wave or additive method, genome is broken into a series of small segment, and add top connection (adaptor) in the both sides of small segment, then carry out the fundamental unit of bridge-type PCR or emulsion-based PCR (emulsion PCR) amplification formation order-checking by joint primer, again according to the partial sequence design common sequencing primer on joint, genomic dna is checked order.The method of current research DNA methylation mainly contains: the methylation analysis method of PCR-based, the methylation analysis method based on restriction enzyme, methylation analysis method and column chromatography etc. based on bisulfite.It is that to methylate the most frequently used be also method the most accurately in research that bisulfite is processed in conjunction with high-flux sequence, bisulfite sequencing (bisulfite genomics sequencing) is to utilize bisulfite that the unmethylated cytosine(Cyt) (C) in genomic dna is modified to uridylic (U), methylated cytosine(Cyt) (C) remains unchanged, in pcr amplification, uridylic (U) will become thymus pyrimidine (T), therefore the site that methylates just becomes a common C/T single base polymorphisms site, wherein the frequency of allelotrope cytosine(Cyt) (C) is the degree of gene methylation, it is large that the method has data volume, the shortcoming that cost is high.In conjunction with methylate DNA co-immunoprecipitation (the Methylated DNA Immunoprecipitation in addition of high-flux sequence, MeDIP) and methylated CpG in conjunction with territory (methyl-CpG binding domain, MBD) column chromatography, although they can well carry out enrichment to being rich in the region that methylates, but the relative homogeneous of coverage, lacks specific aim.
Sequence capturing technology is a kind of technology of genome specific region being carried out to selective enrichment, it separates interested region by suitable method from genome, and then checked order in this target area, this at low cost targetedly genomics research have very important meaning.
(Brenet F et al.2011 is found in research recently; Harder A et al.2010; Suzuki MM et al.2008): exon methylate recognize before density ratio and expect more general, and first exon and First Intron methylate and distribute greatlyr with the exon in downstream and the intron distributional difference that methylates, the methylated degree of downstream area is not closely related with genetic expression mostly.In a word, transcription initiation site around methylates, first exon region methylate with the relation of gene silencing than upstream promoter region methylate must be related to it more close.Analysis exon methylates the research of genetic expression is of great importance.Existing research is after genome bisulfite is processed mostly, according to C → T conversion, designs the probe of specific target area, the methylated fragment in acquisition target region.This method was carried out bisulfite processing by genome before sequence capturing, had increased the difficulty of probe design and had reduced the efficiency of sequence capturing, the coverage in object region, thereby having limited the popularity of applying.
Summary of the invention
For the problem existing in the above-mentioned research method that methylates based on bisulfite, the present invention proposes a kind of binding sequence and catch the method checking order with bisulfite, the method can be caught the target area in several samples by a sequence capturing test, evaluating objects region methylation state accurately and effectively, reduce the difficulty of probe design, increase the feasibility of operation and application, making that the high-precision detection that methylates of high-throughput is carried out in interested target sequence and region in full genome becomes a reality, and the method is pointed strong and the cost-saving and feature time also.
The object region the present invention relates to is exon region, exon group is caught with bisulfite order-checking and combined, the distribution situation that methylates that detects the full genome exon of mankind group with this, this finds that to research exon is with a wide range of applications to the effect of gene expression regulation.The main flow process of this technology: first genomic dna is interrupted at random, add after given joint and carry out sequence capturing by solution hybridization method, catch in the DNA that gets off and add foreign DNA and then carry out bisulfite processing, the DNA handling well is carried out to pcr amplification, TA clone detects bisulfite treatment effect, then library is carried out detection by quantitative and carried out high-flux sequence with new-generation sequencing instrument, finally carry out high-precision methylation status analysis within the scope of specific regions.This technical scheme is mainly made up of following five parts: selection, library preparation, sequence capturing, bisulfite processing, the order-checking of upper machine and the data analysis of sequence capturing probe.
The technical program middle probe simplicity of design, can be based on current liquid phase or solid phase chip hybridization technique designing probe, probe length can be from 60mer-120mer not etc.As select probe SureSelect Human All Exon 38M kit (Agilent), this probe to cover the full exon region of people and part miRNA region, with a wherein chain complementation in genome object region, mean length is 120mer.
The fragmentation of step 1 sample gene group DNA and foreign gene group DNA
Initial object research material and can be any species (for example people as the material of foreign gene group DNA, plant, insect) genomic dna, utilize physics or chemical process genomic dna to be interrupted to the fragment into 200-300bp as ultrasonic wave fragmentation method.Foreign gene group DNA preferably selects the λ DNA that does not methylate and modify; its effect be in the time that bisulfite is processed together with sample efficient co-processing; micro-DNA fragmentation is played a protective role, reduce to greatest extent the destruction of bisulfite to minim DNA.
Get and there is no the complete genome group DNA approximately 5 μ g of the pollution such as albumen, RNA and the λ DNA as foreign gene group, by ultrasonic fragmentation method, genomic dna is interrupted to the fragment into big or small 200-300bp.
DNA after interrupting at random reclaims after purifying, end reparation is carried out in effect by enzymes such as T4DNA polysaccharase (T4DNAPolymerase), Klenow fragment (Klenow Fragment) and T4 polynucleotide kinases (T4Polynucleotide Kinase), forms the DNA fragmentation of flat end.Then utilize Klenow fragment (Klenow Frgment) (3 '-5 ' exo-) polysaccharase and dATP to add " A " base at 3 ' end of the sequence filling.
The sequence that 3 ' end adds " A " base under the effect of T4DNA ligase enzyme (T4DNA Ligase) with particular design and the joint modified of methylating (C site methylate modification) be connected, purifying reclaims, as reclaimed after the DNA in purification reaction system with MiniElute PCR Purification Kit (Qiagen), it is carried out quantitatively, as adopted Qubit (Invitrogen) method etc.
Get 500-1000ng connection product library and carry out liquid phase or solid-phase hybridization, as adopt Agilent solution hybridization platform or Nimblegen solid phase or solution hybridization platform to carry out, after adding tab closure sequence to wait to hybridize in hybridization system simultaneously, collect sequence the purifying of catching by methods such as sex change, obtain the DNA molecular with hybridization probe regional complementarity.
By catch the DNA getting off add 200ng fragmentation λ DNA, then one reinstate bisulfite processing, as utilize EZ DNA Methylation-Gold Kit
tM(ZYMO) make non-methylated cytosine be converted to uridylic.
Glue purification is cut in step 4 pcr amplification and library
DNA after changing with bisulfite is template, add PCR primer sequence, use for the warm start taq enzyme of the DNA after bisulfite conversion and carry out pcr amplification (r-taq of available routine or other polymeric enzymatic amplification), amplified production can carry out purifying by for example following three kinds of methods, is respectively magnetic beads for purifying, purification column purifying, 2% agarose gel electrophoresis purifying.It is quantitative that the product that purifying reclaims carries out QPCR, then treats machine order-checking.
By the sequence of catching after bisulfite is processed at s-generation order-checking platform, for example on Solexa order-checking platform, adopt the method checking order while synthesize to carry out sequencing.In order to make a distinction after order-checking deriving from DNA library prepared by different samples, the sequence label of 6bp or 8bp is incorporated into a side of fragment by joint or PCR primer, upper machine order-checking after can conveniently different libraries directly being mixed like this.When analytical data, the sequence of reference is hg18 (known whole genome sequence)
The analysis process of the raw data of order-checking gained is with reference to LI Y et al., Nature (2008) (JWang, et al., (2008) .The DNA Methylome of Human Peripheral Blood Mononuclear Cells.Nature, 456:60.), in actual applications, the selection meeting of reference data is along with studied genome source is different and different, fundamental analysis process comprises following key step: the C of the data normal chain that order-checking is obtained all changes into T, the G of complementary strand all changes into A, use SOAP program (Li R, Li Y., Kristiansen K. & Wang, J. (2008) .SOAP:short oligonucleotide alignment program.Bioinformatics, 24:713-714.) compare and be all converted into T to C respectively, G is all converted into the hg18 of A with reference on genome, wherein allow the mispairing of two bases.Then statistics comparison is in the section of reading (reads) of unique position, target area, and compare the above section of reading of reference sequences with these, filter as filtration parameter using Depth > 4, using the information that methylates in site by after filtering as the actual information that methylates measuring, and take carry out follow-up comparison information analysis for basic.
The sequence measurement that carries out again sequence capturing from the bisulfite processing postgenome of conventional study report is different, in the present invention, first carries out the sequence capturing of specific region, uses afterwards sulfiting again.Therefore, in the process of sequence capturing, the methylated state of sample does not change, and just does not need to consider the impact of object region methylation level on sequence capturing efficiency when design capture probe.Can catch all target areas and verify its situation that methylates, distribute to come design chips and needn't forethought methylate again, the data of acquisition are truer and fully.The present invention can make the probe design method in sequence capturing comparatively simple, and capture rate is high, so can be just analyzing gene group methylation level comprehensively and exactly.
The technical program middle probe simplicity of design, can be based on current liquid phase or solid phase chip hybridization technique designing probe, probe length can be from 60-120mer not etc.
This technology is in the time of library construction, the connection of label (Index) joint methylates before sequence capturing, can, by carry out sequence capturing after several sample mix simultaneously, greatly provide cost savings like this, thus realize large sample amount high-throughput methylation level detect.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.
One aspect of the present invention provides the one high-flux detection method that methylates, it comprises the following steps: the selection of sequence capturing probe, library preparation, sequence capturing, bisulfite processing, the order-checking of upper machine and data analysis, wherein sequence capturing and bisulfite are processed the joint Connection Step prepared in library and pcr amplification and library and cut between glue purification step and carry out.
In a specific embodiment of the present invention, the described probe methylating in high-flux detection method can be the probe for liquid phase or solid phase chip hybridization, preferably the length of described probe is 60mer-120mer, and more preferably the mean length of described probe is 120mer.
In a specific embodiment of the present invention, the described library preparation process methylating in high-flux detection method comprises the connection of the label joint that methylates.
In a specific embodiment of the present invention, in the high-flux detection method that methylates, before sequence capturing, carry out following library preparation process:
The fragmentation of step 1 sample gene group DNA and foreign gene group DNA
Sample gene group DNA and can be any species as foreign gene group DNA, includes but not limited to the genomic dna of people, plant, insect; Utilize physics or chemical process, preferred ultrasonic wave fragmentation method interrupts preferred 5 μ g genomic dnas at random, preferably interrupts the fragment into 200-300bp; Preferably foreign gene group DNA is the λ DNA that does not methylate and modify;
DNA after interrupting at random reclaims after purifying, carries out end reparation by the effect of preferred T4DNA polysaccharase, Klenow fragment and T4 polynucleotide kinase enzyme, forms the DNA fragmentation of flat end; Then preferably utilize Klenow fragment (3 '-5 ' exo-) polysaccharase and dATP to add " A " base at 3 ' end of the sequence filling;
The methylate connection of joint of step 3 PEI
3 ' end adds that the sequence of " A " base is at ligase enzyme, under the effect of preferred T4DNA ligase enzyme with methylate modifications, the preferred C site joint of modifying that methylates connects, purifying recovery, carries out quantitatively it.
In a specific embodiment of the present invention, the PEI described in high-flux detection method that the methylates joint that methylates comprises:
The PE Index_ adapter 1 that methylates:
Phos/
tCAAGTaGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (SEQ ID NO:1, all C site methylate modification), and
The PE Index_ adapter 2 that methylates:
TACACTCTTTCCCTACACGACGCTCTTCCGATCTA
CTTGAT(SEQ ID NO：2)。
Can change in the present invention the base of underscore part in above-mentioned joint sequence, thereby produce more, different Tag primers, for multiple different sample.
In a specific embodiment of the present invention, the sequence capturing step methylating in high-flux detection method comprises the connection product library in the preparation process of library at solid phase or solution hybridization platform, preferably solution hybridization platform is hybridized, wherein preferably use 500-1000ng to connect the hybridization of product library, in hybridization system, add tab closure sequence simultaneously; After waiting to hybridize, collect sequence the purifying of catching by methods such as sex change, obtain and the DNA molecular of hybridization probe regional complementarity, wherein preferably externally aobvious subgroup is caught.
In a specific embodiment of the present invention, the tab closure sequence using in the sequence capturing step methylating in high-flux detection method is the PEI PEI that the uses complementary sequence of joint that methylates that methylates in the Connection Step of joint, and is selected from
The PE Index_ adapter 1 that methylates:
Phos/TCAAGTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (SEQ ID NO:1), or
The PE Index_ adapter 2 that methylates:
The complementary sequence of TACACTCTTTCCCTACACGACGCTCTTCCGATCTACTTGAT (SEQ ID NO:2), all C site of wherein said joint is all through the modification that methylates.
In a specific embodiment of the present invention, the bisulfite treatment step methylating in high-flux detection method comprises outside the pale of civilization the DNA fragmentation obtaining in sequence capturing step source genomic dna, one reinstates bisulfite processes, and foreign gene group DNA preferably 200ng through the λ of fragmentation DNA.
The present invention further provides the one high-flux detection method that methylates, wherein after bisulfite is processed, carried out the step that glue purification is cut in pcr amplification in the preparation of following library and library:
DNA after changing with bisulfite is template, add PCR primer sequence to carry out pcr amplification, the polysaccharase that wherein used comprises for the warm start taq enzyme of the DNA after bisulfite conversion, conventional r-taq or other polymeric enzymatic amplification, the warm start taq enzyme of the DNA after preferably changing for bisulfite;
Amplified production is carried out to purifying, and purification process includes but not limited to magnetic beads for purifying, purification column purifying, 2% agarose gel electrophoresis purifying; It is quantitative that the product that purifying reclaims carries out QPCR, then treats machine order-checking.
In a specific embodiment of the present invention, pcr amplification step further comprises a side that the sequence label of preferred 6bp or 8bp is incorporated into DNA fragmentation by joint or PCR primer.
In a specific embodiment of the present invention, methylating, the PCR primer sequence using in root pcr amplification in high-flux detection method comprises:
The public primer of P1:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(SEQ ID NO：3)，
Index1 primer:
CAAGCAGAAGACGGCATACGAGAT
cTTGATgTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:4), and
CAAGCAGAAGAC GGCATAC GAGAT
TCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO：5)。
Wherein can change the base of underscore part in above-mentioned PCR primer, thereby produce more, different Tag primers, for multiple different sample.
In a specific embodiment of the present invention, the upper machine order-checking and the data analysis that methylate in high-flux detection method comprise that the sequence that bisulfite treatment step is obtained is at order-checking platform, preferably s-generation order-checking platform, on preferred Solexa order-checking platform, check order, the line number of going forward side by side compares according to one's analysis.
The sequencing library that the present invention provides the high-flux detection method that methylates according to the present invention to build on the other hand.
The present invention provides the purposes for the high-flux sequence that methylates according to the sequencing library of the high-flux detection method structure that methylates according to the present invention on the other hand.
Further, the high-flux detection method that methylates according to the present invention is applicable to exon group order-checking, further preferably for detection of the distribution situation that methylates of the full genome exon of mankind group.
Accompanying drawing explanation
Fig. 1: MDC PCR 2100 detected results.
Fig. 2: YH PCR 2100 detected results.
Fig. 3: YH and the MDC coverage on every karyomit(e) distributes.Wherein red post YH represents YH Whole Blood Genomic DNA, green post mDC (Mature dendritic cells) represents ripe dendritic cell DNA, X-coordinate (chromosome) represents karyomit(e) number, and ordinate zou (coverage of ech chromosome) represents order-checking coverage.
Fig. 4: YH and MDC be depth profile on every karyomit(e).Its Green post YH represents YH Whole Blood Genomic DNA, and red post mDC represents ripe dendritic cell DNA, and X-coordinate (chromosome) represents karyomit(e) number, and ordinate zou (depth of ech chromosome) represents the order-checking degree of depth.
Fig. 5: YH MDC catches the distribution of fragment on gene.
Fig. 6: MDC catches target area and full genome bisulfite data comparison.X-coordinate and ordinate zou represent respectively methyl rate (methylation rate in target region) that target acquistion draws and the methyl rate of genome sequencing.Pearson Correlation represents Pearson correlation coefficient.
Fig. 7: YH catches target area and full genome bisulfite data comparison.X-coordinate and ordinate zou represent respectively methyl rate (methylation rate in target region) that target acquistion draws and the methyl rate of genome sequencing.Pearson Correlation represents Pearson correlation coefficient.
Embodiment
Main laboratory apparatus list
Related reagent list
Sequence list (5 '-> 3 ', what underscore part represented is label)
DNA segment:
Use covaris-S2 to interrupt instrument, YH Whole Blood Genomic DNA (Yan Di and Huang Di, two legendary rulers of remote antiquity's Whole Blood Genomic DNA, the genomic dna extracting from only one China man blood) and MDC DNA (people's immunocyte is DNA) sample respectively interrupt (external source λ DNA is also like this) take 5 μ g as initial amount.According to following parameter setting:
| Treatment1 (processing 1) | Duty/cycle (%) (duty ratio) | 10 |
| Intensity (intensity) | 5 | |
| Cycle/burst (circulation/pulse) | 200 | |
| Time (min) (time (second)) | 50 | |
| Treatment2 (processing 2) | Time (s) (time (second)) | 0 |
| Treatment (processing 3) | Time (s) (time (second)) | 0 |
| Treatment4 (processing 4) | Time (s) (time (second)) | 0 |
| Cycles (circulation) | 3 |
After 2% agarose gel electrophoresis detection is qualified, (DNA fragmentation master tape concentrates between 200-300bp sample after interrupting, pollute without albumen, RNA), through QIAquick PCRPurification Kit (Qiagen) purifying, back dissolving is in the Elution Buffer (EB) of 32 μ l.
Genomic dna end modified:
DNA according to the form below obtained in the previous step is prepared to end in the centrifuge tube of 1.5ml and repairs reaction system:
Then centrifuge tube is put into the upper reaction of Thermomixer (Eppendrf) 30min that is adjusted to 20 ℃.React rear and carried out purifying with QIAquick PCR Purification Kit (Qiagen), finally sample has been dissolved in 34 μ l Elution Buffer (EB).
DNA fragmentation 3 ' end adds " A " base:
DNA according to the form below obtained in the previous step is prepared and is added " A " reaction system in the centrifuge tube of 1.5ml:
Then centrifuge tube is put into the upper reaction of Thermomixer (Eppendrf) 30min that is adjusted to 37 ℃.React rear and carried out purifying with MiniElute PCR Purification Kit (Qiagen), finally sample has been dissolved in to 16 μ L Elution Buffer (EB).
The methylate connection of joint of PEI:
DNA according to the form below obtained in the previous step is prepared to the joint ligation system that methylates:
Then be put into the upper reaction of Thermomixer (Eppendrf) 15min that is adjusted to 20 ℃.React rear and carried out purifying with MiniElute PCR Purification Kit (Qiagen), sample has been dissolved in to 12 μ L Elution Buffer (EB).Then adopt Qubit (Invitrogen) to carry out concentration detection, according to concentration detected result, the concentration of DNA is controlled to 147ng/ul (by concentrated or dilution mode).
Sequence capturing:
Take a hybridization (reagent is all from SureSelect Human All Exon 38Mkit) as example:
A. the preparation of hybridizing reagent A:
The preparation (C) of b.SureSelect Oligo Library Mix:
In PCR pipe, after packing 5 μ L Oligo Capture Library, add RNase Block (the wherein RNase Block: nuclease-free water=1: 3), be placed on ice after 2 μ L dilutions.
C. the configuration of library and block mixed liquid B:
| Library | 3.4μL 147ng/μL |
| |
2.5μL |
| |
2.5μL |
| Index Block | 0.6μL |
| Amount to | 9μL |
The PEI joint that methylates is double-stranded DNA, while wherein using PE-index to methylate Adapter1 strand (SEQ ID NO:1), seal accordingly sequence (Index Block) and be the methylate complementary sequence of Adapter1 strand (SEQID NO:1) of PE-index in above-mentioned PEI methylates the Connection Step of joint; While using PE-index to methylate Adapter2 strand (SEQ ID NO:2) in above-mentioned PEI methylates the Connection Step of joint, the IndexBlock adding is the methylate complementary sequence of Adapter2 strand (SEQ ID NO:2) of PE-index.
In PCR pipe, configure mixed liquid B, and mixed solution mixed with pipettor, cover tightly pipe lid, be placed in PCR instrument by following condition operation thermal cycler program:
D. hybridizing reagent A is placed in thermal cycler and is at least kept 5 minutes in 65 ℃.
E. SureSelect Oligo Library Mix (C) is placed in thermal cycler and is at least kept 2 minutes in 65 ℃.
F. keep PCR pipe in 65 ℃, with 20 μ l pipettors, the Hybridization BufferA of 13 μ l is transferred in SureSelect Oligo Library Mix (C) pipe rapidly; Then with 20ul pipettor, the mixed solution in library and block mixed liquid B is all transferred in SureSelect Oligo Library Mix (C) pipe rapidly, with pipettor slowly up and down inhale blow 8 to 10 times.
G. cover tightly PCR pipe lid, in 65 ℃ of (thermal cycler heat lid is made as 105 ℃) hybridization 24 hours.
Sample wash-out (reagent is all from SureSelect Human All Exon 38M kit):
A. magnetic bead is prepared
Each hybridization is got 50 μ l DynabeadsM-280Streptavidin magnetic beads in a new 1.5mL centrifuge tube;
Use 200 μ L SureSelect Binding buffer to wash three times;
Finally add the resuspended magnetic bead of 200 μ l SureSelect Binding buffer.
B. elution samples
By be equipped with hybrid mixed liquid and magnetic bead centrifuge tube symmetry be fixed on BD Clay AdamsNutator Mixer or similarly upper 360 degree of device rotate and mix, under room temperature, hatch 30 minutes, instantaneous centrifugal 3s is placed on and on magnetic frame, removes supernatant;
With the resuspended magnetic bead of 500 μ l SureSelect Wash Buffer# 1, and on whirlpool mixed instrument, vibrate and within 5 seconds, mix sample, under room temperature, hatch sample 15 minutes;
Wash according to the following steps magnetic bead and obtain object fragment:
A) centrifuge tube is shifted and is placed on magnetic frame, leave standstill 5-10min to clarification, and remove supernatant liquor as far as possible;
B) with the resuspended magnetic bead of 500 μ l SureSelect Wash Buffer# 2, and on whirlpool mixed instrument, vibrate 5 seconds to mix sample;
C) sample is put in Thermomixer to 65 ℃ and hatched 10 minutes, put upside down centrifuge tube to mix sample, instantaneous centrifugal 3 seconds;
D) repeat " step a is to step c " 2 times, Wash Buffer# 2 is removed clean as far as possible.
E) add 50 μ L SureSelect Elution Buffer, and on whirlpool mixed instrument, vibrate 5 seconds with resuspended magnetic bead, sample is put under room temperature and is hatched 10 minutes;
F) centrifuge tube is shifted and is placed on Dynal magnetic frame, leave standstill 5-10min to clarification;
G) with pipettor, the elutriant that contains sample is transferred in a new 1.5ml centrifuge tube;
H) in the DNA catching, add 50 μ l SureSelect Neutralization Buffer.
I) use 1.8 times of Ampure Beads to remove the salinity of catching in sample solution.
Catching product adds foreign DNA to carry out bisulfite co-processing:
Catch the external source λ DNA that adds 200ng fragmentation in product, then add bisulfite to process 2h.Bisulfite is processed and is adopted EZ DNA Methylation-Gold Kit
tM(ZYMO), concrete steps are as follows:
A) preparation of CT Conversion Reagent: take out CTConversion Reagent (solid mixture) from test kit test kit, then add water, the M-Dissolving Buffer of 50 μ l and the M-Dilution Buffer of 300 μ l of 900 μ l in the CTConversion Reagent of a pipe.At room temperature dissolve and shake 10 minutes or on shaking table, shake 10 minutes.
B) preparation of M-WASH BUFFER: add the ethanol of 24ml 100% and prepare final operable M-Wash Buffer in M-WashBuffer, make hook (using marking pen mark) after adding ethanol on lid, show to use.
C) DNA to be converted is installed in PCR pipe to moisturizing to 20 μ l according to dividing.
D) in PCR pipe, add the CT Conversion Reagent of 130 μ l, by flicking test tube or pipettor operates biased sample.
E) sample hose is put into operation according to the following steps on PCR instrument:
Place 2.5 hours for 64 ℃
Carry out at once next step operation or storage (maximum 20 hours) at 4 ℃.
F) the M-Binding Buffer that adds 600 μ l is to Zymo-Spin IC
tMin Column, and post is put in the Collection Tube providing as test kit.
G) filling sample is to Zymo-Spin IC
tMcolumn contains M-Binding Buffer.Close the lid post is put upside down and carried out biased sample for several times.
H) (> 10,000xg) centrifugal 30 seconds at full speed, removes effluent liquid.
I) add the M-Wash Buffer of 200 μ l in post, centrifugal 30 seconds at full speed.
J) the M-Desulphonation Buffer that adds 200 μ l places 15 minutes in post and under room temperature (20 ℃-30 ℃), after cultivation, and centrifugal 30 seconds at full speed.
K) add the M-Wash Buffer of 200 μ l in post, centrifugal 30 seconds at full speed; Add again the M-Wash Buffer of 200 μ l and centrifugal 30 seconds.
L) the M-Elution Buffer that directly adds 10 μ l is in base for post matter.Post is placed in the pipe of 1.5ml, at full speed centrifugal come eluted dna.
Pcr amplification and library size are selected: DNA obtained in the previous step is prepared to PCR reaction system by following system:
| The DNA of bisulfite processing | 10μl |
| dNTP(2.5mM) | 2μl |
| 10x pcr buffer | 2.5ul |
| JumpStartTM Taq DNA Polymerase | 0.3μl |
| The public primer of P1 | 0.5μl |
| Index1 or 2 primers | 0.5μl |
| dH 2O | 9.2μl |
| Cumulative volume | 25μl |
PCR reaction conditions:
Amplified production is carried out to purifying with PCR Purification Kit (Qiagen), then carry out electrophoresis with 2% agarose gel, then glue being cut in object size library selects, adopt MiniElute PCR Purification Kit (Qiagen) to carry out glue purification recovery, finally library is dissolved in 20 μ l EB.
Library detection: TA clone detects sulphite to genomic efficiency of conversion; Agilent 2100Bioanalyzer detects library output; QPCR detection by quantitative library output.
Order-checking and data analysis:
Sample is carried out to two end sequencings in Solexa order-checking platform.By with the result of the full genome sulphite order-checking in same sample source and the data analysis comparison that full exon sequence is caught, analytical sequence is caught the feasibility of methylation analysis method.
Experimental result:
The Agilent 2100Bioanalyzer detected result of 1PCR product
The PCR product Agilent 2100Bioanalyzer detected result of Fig. 1 and Fig. 2 shows that the initial YH of 5 μ g (Yan Di and Huang Di, two legendary rulers of remote antiquity's Whole Blood Genomic DNA) and MDC DNA (ripe dendritic cell DNA) can build and utilize high-throughput new-generation sequencing instrument to carry out the library that methylates of high-flux sequence, show that in conjunction with actual sequencing data analytical results hereinafter described this inventive method is practical, can be applicable in practical study.
2TA clones result
Table 1YH and MDC DNA build library text storehouse quality examination result
2 libraries are selected respectively 36 and 41 clones and are carried out quality examination, and results conversion efficiency all, more than 99%, illustrates that bisulfite processing has realized efficient conversion.
3 data analyses
3.1 with full genome alignment rate comparison
Table 2YH, MDC and routine are built storehouse sequencing data result comparison result
As can be seen from the above table, the comparison rate of YH and MDC is respectively 91.80% and 87.17%.Comparison rate, the transformation efficiency of capture rate and BS is all in normal range.
The distribution plan of 3.2 coverages
Table 3YH, MDC coverage distribution plan
The distribution situation of table 3 display data under the different multiplier covering, in very concentrated being distributed in target area of current order-checking amount data.
3.2.1 the average coverage of each karyomit(e) distributes and checks order depth profile as can be seen from Figure 3, and sequencing data can cover the target area on every karyomit(e) substantially.
Fig. 4 is presented in target area, and the degree of depth on every karyomit(e) is all in 50 × left and right, and the comparatively accurate site information that methylates can be provided in the process of calculating methyl rate.
What Fig. 5 showed is the distributions on karyomit(e) of the data that capture on No. 12 karyomit(e)s, can find out that data only have enrichment in the place that has target area.
3.3 data dependence comparisons
Fig. 6 and Fig. 7 be respectively two samples (MDC and YH) use the sequencing data that obtains of the inventive method methylate correlation analysis with use full genome normally to build storehouse to obtain the methylate comparison of correlation analysis of corresponding sequencing data, data are filter depth 10 × the methyl rate distribution situation of above data; Can see that the methyl rate on same loci is basically identical, the relation conefficient of MDC sample is 0.94 (Pearson correlation coefficient), and the relation conefficient of YH sample is 0.93.
By being carried out to specific region, YH and MDC catch library construction, obtain sequencing data and conventional library order-checking corresponding data and carry out information analysis comparative result, from comparison efficiency, coverage, every chromosomal methyl rate and dependency, each side fraction of coverage is all good, methyl rate also has good consistence, and technique can reach very dark data volume among a small circle, these have all illustrated that the methylate high-flux sequence research of employing specific region is practicable, this method reduces the difficulty of probe design, increase the feasibility of operation and application, making that the high-precision detection that methylates of high-throughput is carried out in interested target sequence and region in full genome becomes a reality, and pointed strong and the cost-saving and feature time.
Reference:
[1]Brena RM，Huang TH，Plass C.Quantitative assessment of DNA methylation：Potential applications for disease diagnosis，classification，and prognosis in clinical settings.J Mol Med.2006May；84(5)：365-77.
[2]Egger G，Liang G，Aparicio A et al.Epigenetics in human disease and prospects for epigenetic therapy.Nature 2004 May27；429(6990)：457-63.
[3]Gu H，Bock C，Mikkelsen TS，et al.Genome-scale DNAmethylation mapping of clinical samples at single-nucleotide resolution.Nat Methods.2010 Feb；7(2)：133-6.
[4]Hodges E，Smith AD，et al.High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing.Genome Res.2009Sep；19(9)：1593-605.
[5]Brenet F，Moh M，et al.DNA methylation of the first exon is tightly linked to transcriptional silencing.PLoS One.2011 Jan18；6(1)：e14524.
[6]Harder A，Titze S，et al.Monozygotic twins with neurofibromatosis type 1(NF1)display differences in methylation of NF1 gene promoter elements，5′untranslated region，exon and intron1.Twin Res Hum Genet.2010Dec；13(6)：582-94.
[7]Suzuki MM，Bird A.DNA methylation landscapes：provocative insights from epigenomics.Nat Rev Genet.2008Jun；9(6)：465-76.
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